The members of the poxvirus family have large double-stranded DNA genomes encoding several hundred proteins (Moss, B. 2007 “Poxviridae: The Viruses and Their Replication” in Fields Virology, 5th Ed. (D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Straus, Eds), Lippincott Williams & Wilkins, Philadelphia, Pa.). Poxviruses are divided into the subfamilies Chordopoavirinae and Entomopaxvirinae, based on vertebrate and insect host range. The subfamily Chordopoxvirinae consists of eight genera: Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus, and Yatapoxvirus. The prototypal member of the genus Orthopoxvirus is vaccinia virus. Vaccinia virus (VACV), the first recombinant virus shown to induce a protective immune response against an unrelated pathogen (Moss, B., G. L. Smith, J. L. Geria, and R. H. Purcell. 1984. Live recombinant vaccinia virus protects chimpanzees against hepatitis B. Nature 311:67-69; Paoletti, E., B. R, Lipinskas, C. Samsonolf S. R. Mercer, and D. Panicali. 1984. Construction of live vaccines using genetically engineered poxviruses; biological activity of vaccinia virus recombinants expressing the hepatitis B virus surface antigen and the herpes simplex virus glycoprotein D. Proc. Natl. Acad. Sci. USA 81:193-197), is being employed as a vector for veterinary and wildlife vaccines (Moss, B. 1996. Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc. Natl. Acad. Sci. USA 93:11341-11348). Development of recombinant VACV for human use, however, has been impeded by safety concerns. For this reason, there is interest in modified VACV Ankara (MVA), a highly attenuated smallpox vaccine with an exemplary safety profile even in immunodeficient animals (Mayr, A., V. Hochstein-Mintzel, and H. Stickl. 1975. Passage history, properties, and applicability of the attenuated vaccinia virus strain MVA. Infection 3:6-14. (In German); Stickl, H., V. Hochstein-Mintzel, A. Mayr, H. C. Huber, H. Schafer, and A. Holzner. 1974. MVA vaccination against smallpox: clinical trial of an attenuated live vaccinia virus strain (MVA). Dtsch. Med. Wschr. 99:2386-2392 (In German); Stittelaar, K. J., T. Kuiken, R. L. de Swart, G. van Amerongen, H. W. Vos, H. G. Niesters, P. van Schalkwijk, T. van der Kwast, L. S. Wyatt, B. Moss, and A. D. Osterhaus. 2001. Safety of modified vaccinia virus Ankara (MVA) in immune-suppressed macaques. Vaccine 19:3700-3709). The genomic sequence of MVA (Mayr, A. et al. 1978 Zentralbl Bakteriol 167:375-390), which cannot grow in most mammalian cells and which is a good candidate for a recombinant vaccine vector, is known (Sutter, G. and Moss, B. 1992 Proc Natl Acad Sci USA 89:10847-10851; and Sutter, G. et al. 1994 Vaccine 12:1032-1040) has been passaged over 570 times in chicken embryo fibroblasts, during which six major deletions relative to the parental wild-type strain Ankara, accompanied by a severe restriction in host range, have occurred (Meyer, H. et al. 1991 J Gen Virol 72:1031-1038). MVA is severely host range restricted and propagates poorly or not at all in most mammalian cells because of a block in virion assembly (Sutter, G., and B. Moss. 1992. Nonreplicating vaccinia vector efficiently expresses recombinant genes. Proc. Natl. Acad. Sci. USA 89:10847-10851). Initial experiments with recombinant MVA (rMVA) demonstrated its ability to robustly express foreign proteins (Sutter, G., and B. Moss. 1992. Nonreplicating vaccinia vector efficiently expresses recombinant genes. Proc. Natl. Acad. Sci. USA 89:10847-10851) and induce protective humoral and cell-mediated immunity (Sutter, G., L. S. Wyatt, P. L. Foley, J. R. Bennink, and B. Moss. 1994. A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus. Vaccine 12:1032-1040). Currently, rMVA candidate vaccines expressing genes from a wide variety of pathogens are undergoing animal and human testing (Gomez, C. E., J. L. Najera, M. Krupa, and M. Esteban. 2008. The poxvirus vectors MVA and NYVAC a gene delivery systems for vaccination against infection diseases and cancer. Curr. Gene Ther. 8:97-120).
While developing candidate human immunodeficiency virus (HIV) and other vaccines, it was observed that mutant rMVA loses the ability to express foreign proteins after tissue culture passage (Stittelaar, K. J., L. S. Wyatt, R. L. de Swart, H. W. Vos, J. Groen, G. van Amerongen, R. S. van Binnendijk, S. Rozenblatt, B. Moss. and A. Osterhaus. 2000. Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of passively acquired antibodies. J. Virol, 74:4236-4243; Wyatt, L. S., I. M. Belyakov, P. L. Earl, J. A. Berzofsky, and B. Moss. 2008. Enhanced cell surface expression, immunogenicity and genetic stability resulting from a spontaneous truncation of HIV Env expressed by a recombinant MVA. Virology 372:260-272; Wyatt, L. S., S. T. Shors, B. R. Murphy, and B. Moss. 1996. Development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model. Vaccine 14:1451-1458). This instability may initially go undetected, however, unless individual plaques are isolated and analyzed. Nevertheless, once established in the population, the nonexpressors can rapidly overgrow the original rMVA. These considerations are particularly important for production of large vaccine seed stocks of rMVA. The instability of cloned genes in MVA is surprising, since MVA had already undergone genetic changes during its adaptation through hundreds of passages in chicken embryo fibroblasts (CEFs) and is now quite stable. Indeed, identical 167,000-bp genome sequences have been reported for three independent plaque isolates, accession numbers U94848, AY603355, and DQ983236, and by Antoine et al. (Antoine, G., F. Scheiflinger, F. Dorner, and F. G. Falkner. 2006. Corrigendum 10 “The complete genomic sequence of the modified vaccinia Ankara (MVA) strain: comparison with other orthopoxviruses.” Virology 350:501-502. [Correction to 244:365, 1998.]). Although the cause of the instability of the gene inserts had not been previously investigated, harmful effects of the recombinant protein seem to play a role in the selective advantage of nonexpressing mutants. Thus, reducing the expression level of parainfluenza virus and measles virus transmembrane proteins and deleting part of the cytoplasmic tail of HIV Env improves the stability of rMVAs (Stittelaar, K. J., L. S. Wyatt, R. L. de Swart, H. W. Vos, J. Groen, G. van Amerongen, R. S. van Binnandijk, S. Rozenblatt, B. Moss. and A. Osterhaus. 2000. Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of passively acquired antibodies. J. Virol, 74:4236-4243; Wyatt, L. S., I. M. Belyakov, P. L. Earl, J. A. Berzofsky, and B. Moss. 2008. Enhanced cell surface expression, immunogenicity and genetic stability resulting from a spontaneous truncation of HIV Env expressed by a recombinant MVA. Virology 372:260-272; Wyatt, L. S., S. T. Shors, B. R. Murphy, and B. Moss. 1996. Development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model. Vaccine 14:1451-1458). Reducing expression, however, can also decrease immunogenicity and therefore may be undesirable (Wyatt, L. S., P. L. Earl, J. Vogt, L. A. Eller, D. Chandran, J. Liu, H. L. Robinson, and B. Moss. 2008. Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccines. Vaccine 26:486-493).
In view of the potential value of rMVA as a vaccine, it is important to understand this pernicious instability problem, and to develop methods for constructing stable, recombinant MVA viruses. Additionally, an understanding of the stability problem might provide insights that have application to other DNA expression vectors. The present invention provides such insights and provides for a solution to the problem of constructing stable, recombinant MVA viruses.